|
| Status |
Public on May 15, 2012 |
| Title |
Gill_tissue_K+3_Rep3_msh5 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
C. cinerea excised gill tissue, 3 hours after karyogamy
|
| Organism |
Coprinopsis cinerea |
| Characteristics |
strain: msh5-22
tissue: gill
timepoint: 3 hrs after karyogamy
|
| Treatment protocol |
no treatments
|
| Growth protocol |
Dikaryotic strains wre grown at 37 C for two days and then moved to 25 C with a cycle of 8 hours dark, 16 hours light.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
At the pertinent timepoint, gill tissue from 7-10 mushrooms was separated from the mushroom stipe, and the veil removed. Gill tissue was immediately frozen in liquid nitrogen. RNA was extracted from all the tissue collected using RNeasy Plant mini-prep kit.
|
| Label |
Alexa fluor 555
|
| Label protocol |
20ug of total RNA were reverse transcribed and labelled with Alexa fluor using the Invitrogen Superscript Indirect labeling kit.
|
| |
|
| Channel 2 |
| Source name |
C. cinerea excised gill tissue, 3 hours after karyogamy
|
| Organism |
Coprinopsis cinerea |
| Characteristics |
strain: Java;5-5 x Java;5-4
tissue: gill
timepoint: 3 hrs after karyogamy
|
| Treatment protocol |
no treatments
|
| Growth protocol |
Dikaryotic strains wre grown at 37 C for two days and then moved to 25 C with a cycle of 8 hours dark, 16 hours light.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
At the pertinent timepoint, gill tissue from 7-10 mushrooms was separated from the mushroom stipe, and the veil removed. Gill tissue was immediately frozen in liquid nitrogen. RNA was extracted from all the tissue collected using RNeasy Plant mini-prep kit.
|
| Label |
Alexa fluor 647
|
| Label protocol |
20ug of total RNA were reverse transcribed and labelled with Alexa fluor using the Invitrogen Superscript Indirect labeling kit.
|
| |
|
| |
| Hybridization protocol |
Microarray slides were blocked prior to hybridisation with 5xSSC, 0.1%SDS, 0.1mg.ml-1 BSA at 42oC for 45 min, after which slides were rinsed twice in room temperature 0.1xSSC for 5 min each, followed by a final 10s rinse in water. Slides were dried by centrifugation for 1min in a Labnet C1303 slide spinner. Arrays were placed in Corning hybridization chambers and mSeries Lifterslips (Erie Scientific) placed over the array grids. Labelled cDNAs were combined in 50μl with 30% deionized formamide (Ambion), 5xSSPE, 0.2% SDS, 1μg.μl-1 tRNA (Invitrogen), 40ng.μl-1 oligo dA (Invitrogen). The hybridization mixture was heated at 100oC for 2min, cooled to 25oC, and applied to the microarray slide. Slides were hybridised for 16 hours at 42oC, then washed for 5 min each in 1xSSC, 0.03%SDS (37oC), then 0.2xSSC (ambient temperature), and finally 0.1xSSC (ambient temperature). Slides were dried by centrifugation, as previously, and scanned immediately.
|
| Scan protocol |
Microarray slides were scanned using a GenePix 4200A scanner (Molecular Devices). PMT settings were adjusted so the dye channels were balanced, with a few saturated spots, to maximize data capture. Spots were identified using GenePix Pro software (Molecular Devices) and manual inspection. Scans were quality assessed using the Basic Hybridization Analysis R script (http://cgb.indiana.edu/downloads/1). Spots were manually flagged to be excluded from analysis if there were areas of poor quality such as scratches or dust. Spots were also flagged for omission, using GenePix software, if they fulfilled any of the following criteria: manually flagged, buffer only spots, spots not found, percentage of saturated pixels in both channels > 3, spot pixels < 40.
|
| Data processing |
Data normalization and filtering were performed using the Bioconductor package (http://www.bioconductor.org/) within the R statistical framework (http://www.r-project.org/). The OLIN package was used for intra-slide normalization and log2 transformation. Data were included in further analysis if two of the four replicates were not flagged. Ratio values were exported for further analysis, with individual flagged ratio values omitted. Statistical significance of changing expression was assessed using the one class function in Significance Analysis of Microarrays (SAM) software
|
| |
|
| Submission date |
May 11, 2012 |
| Last update date |
May 15, 2012 |
| Contact name |
Claire Burns |
| E-mail(s) |
burnsc@indiana.edu
|
| Organization name |
Indiana University
|
| Department |
Biology
|
| Lab |
Zolan Lab
|
| Street address |
1001 E 3rd St
|
| City |
Bloomington |
| State/province |
IN |
| ZIP/Postal code |
47404 |
| Country |
USA |
| |
|
| Platform ID |
GPL7697 |
| Series (2) |
| GSE37943 |
Comparison of gene expression during meiosis between wild-type and msh5-deficient strains of Coprinopsis cinerea |
| GSE37968 |
omparison of gene expression during meiosis between wild-type and deficient strains of Coprinopsis cinerea |
|
